Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany.
Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany
J Virol. 2019 Aug 28;93(18). doi: 10.1128/JVI.00679-19. Print 2019 Sep 15.
For members of the , it is known that, besides the structural proteins, nonstructural (NS) proteins also play a critical role in virion formation. Pestiviruses, such as bovine viral diarrhea virus (BVDV), rely on uncleaved NS2-3 for virion formation, while its cleavage product, NS3, is selectively active in RNA replication. This dogma was recently challenged by the selection of gain-of-function mutations in NS2 and NS3 which allowed virion formation in the absence of uncleaved NS2-3 in BVDV type 1 (BVDV-1) variants encoding either a ubiquitin (Ubi) (NS2-Ubi-NS3) or an internal ribosome entry site (IRES) (NS2-IRES-NS3) between NS2 and NS3. To determine whether the ability to adapt to NS2-3-independent virion morphogenesis is conserved among pestiviruses, we studied the corresponding NS2 and NS3 mutations (2/T444-V and 3/M132-A) in classical swine fever virus (CSFV). We observed that these mutations were capable of restoring low-level NS2-3-independent virion formation only for CSFV NS2-Ubi-NS3. Interestingly, a second NS2 mutation (V439-D), identified by selection, was essential for high-titer virion production. Similar to previous findings for BVDV-1, these mutations in NS2 and NS3 allowed for low-titer virion production only in CSFV NS2-IRES-NS3. For efficient virion morphogenesis, additional exchanges in NS4A (A48-T) and NS5B (D280-G) were required, indicating that these proteins cooperate in NS2-3-independent virion formation. Interestingly, both NS5B mutations, selected independently for NS2-IRES-NS3 variants of BVDV-1 and CSFV, are located in the fingertip region of the viral RNA-dependent RNA polymerase, classifying this structural element as a novel determinant for pestiviral NS2-3-independent virion formation. Together, these findings will stimulate further mechanistic studies on the genome packaging of pestiviruses. For members, the nonstructural proteins are essential for virion formation and thus exert a dual role in RNA replication and virion morphogenesis. However, it remains unclear how these proteins are functionalized for either process. In wild-type pestiviruses, the NS3/4A complex is selectively active in RNA replication, while NS2-3/4A is essential for virion formation. Mutations recently identified in BVDV-1 rendered NS3/4A capable of supporting NS2-3-independent virion morphogenesis. A comparison of NS3/4A complexes incapable/capable of supporting virion morphogenesis revealed that changes in NS3/NS4A surface interactions are decisive for the gain of function. However, so far, the role of the NS2 mutations as well as the accessory mutations additionally required in the NS2-IRES-NS3 virus variant has not been clarified. To unravel the course of genome packaging, the additional sets of mutations obtained for a second pestivirus species (CSFV) are of significant importance to develop mechanistic models for this complex process.
对于成员来说，除了结构蛋白外，非结构（NS）蛋白在病毒粒子形成中也起着关键作用。瘟病毒，如牛病毒性腹泻病毒（BVDV），依赖于未切割的 NS2-3 形成病毒粒子，而其切割产物 NS3 则选择性地在 RNA 复制中活跃。这个观点最近受到了 NS2 和 NS3 的功能获得性突变的挑战，这些突变允许 BVDV-1 变体中的 NS2-3 独立病毒粒子形成，这些变体在 NS2 和 NS3 之间编码泛素（Ubi）（NS2-Ubi-NS3）或内部核糖体进入位点（IRES）（NS2-IRES-NS3）。为了确定在瘟病毒中是否存在 NS2-3 独立的病毒粒子形态发生的适应性能力，我们研究了经典猪瘟病毒（CSFV）中相应的 NS2 和 NS3 突变（2/T444-V 和 3/M132-A）。我们观察到，这些突变仅能恢复 CSFV NS2-Ubi-NS3 的低水平 NS2-3 独立病毒粒子形成。有趣的是，通过选择鉴定的第二个 NS2 突变（V439-D）对于高滴度病毒粒子的产生是必需的。与 BVDV-1 的先前发现类似，这些 NS2 和 NS3 中的突变仅允许 CSFV NS2-IRES-NS3 中低滴度病毒粒子的产生。为了实现有效的病毒粒子形态发生，还需要 NS4A（A48-T）和 NS5B（D280-G）中的额外交换，表明这些蛋白在 NS2-3 独立的病毒粒子形成中协作。有趣的是，为 BVDV-1 的 NS2-IRES-NS3 变体独立选择的两个 NS5B 突变均位于病毒 RNA 依赖性 RNA 聚合酶的指尖区域，将该结构元件归类为瘟病毒 NS2-3 独立病毒粒子形成的新型决定因素。这些发现将共同刺激对瘟病毒基因组包装的进一步机制研究。对于成员来说，非结构蛋白对于病毒粒子的形成是必不可少的，因此在 RNA 复制和病毒粒子形态发生中发挥双重作用。然而，这些蛋白如何被功能化以适应任一过程仍不清楚。在野生型瘟病毒中，NS3/4A 复合物在 RNA 复制中选择性地活跃，而 NS2-3/4A 对于病毒粒子的形成是必需的。最近在 BVDV-1 中鉴定的突变使 NS3/4A 能够支持 NS2-3 独立的病毒粒子形态发生。比较不能/能够支持病毒粒子形态发生的 NS3/4A 复合物发现，NS3/NS4A 表面相互作用的变化对于获得功能是决定性的。然而，到目前为止，NS2 突变的作用以及 NS2-IRES-NS3 病毒变体中另外需要的辅助突变尚未阐明。为了揭示基因组包装的过程，对于第二种瘟病毒（CSFV）获得的额外突变集对于开发该复杂过程的机制模型具有重要意义。
